26/09/2022

Taller de Formación Avanzada en Bioseguridad 2022

Tema 5. Bioética: uso de muestras humanas y

de animales de experimentación.

Lluís Montoliu Septiembre 2022

Vicedirector del CNB
Investigador Científico en CNB‐CSIC y CIBERER‐ISCIII
Ex‐Presidente del Comité de Ética del CSIC
Asesor de Presidencia del CSIC en temas de Ética y Science Europe
Miembro del Panel de Ética de ERC

Definitions
• Moral/morality: what people believe to be
right/good or wrong/bad. Set of codes of conduct
adopted governing human behavior.

• Ethics: is a critical reflection about morality. Provides

a systematic way to work through dilemas to
determine the best course of action among
conflicting choices, a generalized conceptual
framework for decision making.

• Bioethics: the study of ethical issues arising from

health care, in biological and medical sciences.

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What we can do in Science What we can do in Science

What we cannot and should not

Research Integrity

EL PAÍS
MATERIA

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Don’t cheat, don’t copy,

¡Cheat, copy, fabricate!
don’t fabricate!
Don’t follow the rules!
Follow the rules!

Why research misconduct is wrong?

• Adds wrong or useless results to the scientific

record
• Wastes resources
• Reduces public trust
• Endangers public funding
• Society: Endangers people’s lives
• Society: Could impact negatively on policy and
legislation

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Research Integrity
• Credibility of science relies on the
quality and reproducibility of
results

• Codes of Good Scientific Practices

2017

Código de Buenas
Prácticas Científicas
del CSIC ‐ 2021

2021

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Compliance with existing rules and laws

• Research with human subjects/human

samples
• Research with animals
• Research with plants/environment
• Use/release of genetically modified
organisms (GMOs)
• Use of biological agents (i.e. viruses)
• Use of databases, privacy
• Intellectual/industrial properties, patents

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The Nagoya Protocol on ABS

DUAL USE
• The Nagoya Protocol on Access and Benefit-sharing
• The Nagoya Protocol on Access to Genetic
Resources and the Fair and Equitable Sharing of
Benefits Arising from their Utilization to the
Convention on Biological Diversity is an international
agreement which aims at sharing the benefits arising
from the utilization of genetic resources in a fair and
equitable way (October 2014)

www.cbd.int/abs/

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MISUSE

1918-1919
H1N1 serotype

~500 Million infected

~50 Million deaths

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Ethics also means… Why caring about Ethics?

• Safety • No Research Project can commence without
• Security Ethics Approval
• Privacy • To ensure full compliance with norms and
• Respect regulations regarding the due respect to
humans, animals, plants and environment
• Well‐being
• To maximize benefits and minimize risks and
• Welfare harms, while considering the needs associated
• Environment with each Research Project

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Why Ethics Approval is important? Comités de ética

• Ethics Approvals are mandatory
•Seres humanos
• Ethics approval protects the researchers and •Animales
the research being conducted •Agentes biológicos de riesgo
•Organismos modificados
• Ethics Approval demonstrates that the
Research Project and the Researchers adhere
genéticamente
to the accepted Ethics standards that are •Integridad científica
expected by the Society

Comités de ética Bioethics

• Nürnberg Code (1947)
•Seres humanos • Human Rights Declaration (1948)
•Animales • Helsinki Declaration (1967)
• Belmont Report (1979)
•Agentes biológicos de riesgo • Comité Consultatif Nationale Bioéthique. France
•Organismos modificados (1983)
genéticamente • …
• National and EU laws
•Integridad científica

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Basic principles in Bioethics
• Principle of non‐maleficence
• Principle of beneficence
• Principle of respect for autonomy
• Principle of justice
Principle of beneficence
• It is our obligation, our duty, to do good to be
of benefit to the human being
• However, it does not mean imposing our
subjective idea of what is good. We must take
into account the freedom and the
authorization from the human being
(informed consent)
26/09/2022
Principle of non‐maleficence
• You should not harm any human being. It
implies a careful consideration and evaluation
of the equilibrium between potential benefits
and risks associated to any medical or
biomedical intervention
Principle of respect for autonomy
• It refers to our due respect to the basic
freedom of every human being to decide on
any intervention, including biomedical issues,
directly affecting him/her (informed consent)
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Principle of justice
• We must ensure the equitative access to Ethics issues in Horizon Europe
potential benefits, biomedical progress and
wellbeing to everyone, to all human beings,
without restrictions o discriminations of any
sort

2017

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Involvement of Human Beings

Does your research involve human participants?
• Volunteers for social/human sciences
• Unable to give informed consent
• Vulnerable individuals or groups
• Children/minors
• Patients
• Healthy volunteers for medical studies
• Physical interventions? Invasive techniques?
Collection of biological samples?

fMRI

Non-invasive
Functional
Magnetic
Resonance

2004/40/CE

0.2  7 Teslas

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Ethics in research involving Informed Consent

human subjects
• Who do we want to protect? The human
subject, the patient
• What do we have to regulate? The use of
human samples (biological and also data) in
research
• We must ensure respect for people and for
human dignity, and a fair distribution of the
benefits and burden of research, while
protecting the values, the rights and interests
of participants
• Introduction, aims of the specific research to be
conducted (plain words)
• Describing the procedures that will be applied
• What type of human biological samples / data will be
taken or used (and the burden associated)
• What needs to be done with biological sample/data upon
finishing the experiment? (keep/anonymize/destroy)
• Benefits and risks of participating
• What, how and who will be contacted concerning results
obtained? (incidental findings, right to know/not‐know)
• Confidentiality, rights for accessing to data and rights to
modify consent
• Right to revoke consent

Comités de ética Spanish Law on Biomedical Research

•Seres humanos LEY 14/2007,

•L14/2007 : CEI y CBE de 3 de julio, de
• RD1090/2015 : CEIm  AEMPS Investigación
biomédica,
publicada en el
B.O.E. el 4 de julio
de 2007

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Spanish Law on Biomedical Research

• principle of privacy
• principle of free autonomy
• principle of traceability
• principle of no discrimitation
• principle of gratuity
Use of Human Cells or Tissues
• Available commercially
• Obtained within the research project
• Obtained from another research Project
• Obtained from a biobank

• Ethics Approvals / Informed Consent

Comités de ética

BioBank
•Seres humanos
•L14/2007 : CEI y CBE
• RD1090/2015 : CEIm  AEMPS
X •RGPD2016 y LOPDGDD 3/2018
Patient/Donor Researcher
•Biobancos RD1716/2011

Collection

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Research involving human subjects HeLa = Henrietta Lacks

• HeLa cells

• Cervical cancer
• 1951 HeLa cells established
• >60.000 scientific publications
• >10.000 patents

Henrietta Lacks (1920-1951)

Research involving human subjects Research involving human subjects

In 2013 Hela cells genome was
released (Landry et al. Genes,
Genomes, Genet.)

The descents of Henrietta Lacks

complained because nobody asked
them permission and they claimed this
was private information

Eventually Henrietta Lacks’ relatives

agreed to release HeLa genome
partially and under strict restrictions

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Comités de ética
•Seres humanos
• L14/2007 : CEI y CBE
• RD1090/2015 : CEIm  AEMPS
• RGPD2016 y LOPDGDD 3/2018
• Biobancos RD1716/2011
• L14/2007 : Comisión de Garantías
para la Donación y Utilización de
Células y Tejidos Humanos (ISCIII)
Protection of Personal Data
Does your research involves personal data
collection and/or processing?
• Sensitive data (religion, ethnicity, politics…)
• Genetic information
• Tracking or observing participants
• Further processing of previously collected
personal data (secondary use)
• Collect only data that are required
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Comisión de Garantías…
• La investigación con células troncales embrionarias humanas.
• Cualquier otra técnica que, utilizando en todo o en parte muestras
biológicas de origen humano, pueda dar lugar a la obtención de células
troncales, incluidas aquellas que pretendan la reprogramación genética
de células no embrionarias para conferirles propiedades de células
troncales embrionarias.
• Cualquier otra línea de investigación que incluya material celular de
origen fetal o embrionario humano u otro funcionalmente semejante.
• La entrada y salida de España de gametos, preembriones, células
embrionarias humanas y líneas celulares provenientes de estas últimas,
células y tejidos fetales, y células o líneas celulares funcionalmente
semejantes a las embriona‐rias con fines de investigación biomédica.
hESC y iPSC
www.eugdpr.org
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Comités de ética
•Seres humanos
•L14/2007 : CEI y CBE
• RD1090/2015 : CEIm  AEMPS
•RGPD2016 y LOPDGDD 3/2018
New GDPR Regulation (EU) 2016/679
• GDPR Breach mandatory in all member states within
72 hours
• Right for data subjects to obtain from the data controller
confirmation as to whether or not personal data
concerning them is being processed, where and for
what purpose. Further, the controller shall provide a
copy of the personal data, free of charge, in an
electronic format. This change is a dramatic shift to data
transparency and empowerment of data subjects.
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New GDPR Regulation (EU) 2016/679
• Replaces Data Protection Directive 95/46/EC
• Approved in April 2016, entered into force 25 May 2018
• The aim of the GDPR is to protect all EU citizens from
privacy and data breaches in an increasingly data-
driven world
• Increased Territorial Scope (extra-territorial
applicability)
• Breach of GDPR can be fined up to 4% of annual
global turnover or €20 Million (whichever is greater)
• Consent: provided in an intelligible and easily
accessible form, using clear and plain language. It must
be as easy to withdraw consent as it is to give it. Opt-in
New GDPR Regulation (EU) 2016/679
• Right to be Forgotten: Data Erasure, the right to be
forgotten entitles the data subject to have the data
controller erase his/her personal data, cease further
dissemination of the data, and potentially have third
parties halt processing of the data
• Data Portability: the right for a data subject to receive
the personal data concerning them, which they have
previously provided in a 'commonly use and machine
readable format' and have the right to transmit that data
to another controller.
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New GDPR Regulation (EU) 2016/679
• Privacy by Design: inclusion of data protection from
the onset of the designing of systems, rather than an
addition. Controllers to hold and process only the data
absolutely necessary for the completion of its duties
(data minimisation), as well as limiting the access to
personal data to those needing to act out the
processing.
• Data Protection Officers: internal record keeping
requirements
Comités de ética
•Seres humanos
•Humanidades y Ciencias Sociales
• Ethics in Social Science and
Humanities (2018)
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New GDPR and Informed Consents
• Informed Consents must contain now:
• the details of the person the participant may contact
at the site should there be a question or the need to
exercise any of the rights detailed in the data privacy
clause.
• the Data Protection Officer at the research institution.
• the express reference to the fact that the participant
may also lodge a complaint with Data protection
authorities should the study participant feel the need
to do so.
Ethics in Social Science and Humanities
• Research participants’ rights are anchored in fundamental human
rights and the fundamental ethical principles that govern all
scientific research.
• Charter of Fundamental Rights of the European Union
• European Convention on Human Rights (ECHR) and its
Protocols
• UN Declaration of Human Rights
• UN Convention on the Rights of Persons with Disabilities
(UN CRPD)
• Nuremberg Code
• Helsinki Declaration
• Belmont Report EC, October 2018
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Ethics in Social Science and Humanities
• The basic ethical principles that have evolved to protect human
participants from harm
• respecting human dignity and integrity
• ensuring honesty and transparency towards research subjects
• respecting individual autonomy and obtaining free and informed
consent (as well as assent whenever relevant)
• protecting vulnerable individuals
• ensuring privacy and confidentiality
• promoting justice and inclusiveness
• minimising harm and maximising benefit
• sharing the benefits with disadvantaged populations, especially if the
research is being carried out in developing countries
• respecting and protecting the environment and future generations
EC, October 2018
Ethics in Social Science and Humanities
• Approval from an Ethics Committee must be
sought prior commencing the research
• Description of the intended research, its
objectives, methods and aims must be provided
• Description of the target group of individuals
which will be the subject of this research and how
these people will be approached (and what type
of consent will be obtained) must be provided
• Provide clear descriptions of your recruitment
policy: who you intend to recruit; where and how
you will reach your target group; your criteria for
inclusion/exclusion EC, October 2018

Ethics in Social Science and Humanities

Comités de ética
• use of deception in research
• covert research •Agentes biológicos de riesgo
• internet research and social media •Organismos modificados
data in research genéticamente (RD178/2004)
• Comisión de Bioseguridad
• Comisión Nacional de Bioseguridad
• Consejo Interministerial de OMGs
EC, October 2018

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Animal Research
Use of animals
• Who do we want to protect? The animals
Does your research involve animals?
• What do we have to regulate? The use of
animals for research and teaching purposes
• Vertebrates • What do we intend to preserve? Animal
• Non‐Human Primates welfare, minimizing any harm, pain or
• Genetically Modified discomfort caused by a given research
• Cloned Farm Animals procedure
• Endangered Species • Which are the underlying principles?

Principles of Animal Research Replacement

• The use of animals for research purposes must be
• The fundamental principles regulating animal avoided if alternative methods not involving
research were described in 1959 by W. M. S. animals are available allowing to undertake such
Russell and R. L. Burch and are commonly experimentation and to reach the same aims
known amd referred as “the 3Rs”
• Researchers must check and review the
• Replacement existence of possible alternative methods
• Reduction allowing to avoid using research animals
unnecessarily
• Refinament
• Researchers must use the “lowest” animal
permiting to reach the expected conclusions
• EU Directive 2010/63/EU

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Replacement

Replacement: in silico comparative genomics

Brain-specific promoter lacZ

lacZ TG1

lacZ TG2

González‐Fernández Á, Bermúdez Silva FJ, López‐Hoyos M, Cobaleda C, Montoliu L, Del Val M, TG3
lacZ
Leech K. Non‐animal‐derived monoclonal antibodies are not ready to substitute current
hybridoma technology. Nat Methods. 2020 Nov;17(11):1069‐1070

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Replacement Reduction
• Researchers must use the minimum number of
animals allowing to reach significant conclusions

• Often wrongly confounded by forcing to use the

smallest possible number of animals

• It refers also to avoid repeating animal experiments

unnecessarily, aiming at obtaining the maximum
number of data from each experiment, sharing and
adequately documenting the results, so that other
colleagues can use them and do not have to repeat
the same experiment

EMMA: current members

CNR/IBC
Istituto di Biologia Cellulare, Monterotondo, Italy

CNRS/CDTA
Centre de Distribution, de Typage et d'Archivage animal, Orléans, France

MRC/MGU
Mammalian Genetics Unit, Harwell, UK

Karolinska Institutet
Karolinska Institutet, Stockholm, Sweden

FCG/IGC
Instituto Gulbenkian de Ciência , Oeiras, Portugal

HMGU/IEG
Institute of Experimental Genetics, Munich, Germany

EMBL/EBI
European Bioinformatics Institute, Hinxton, UK

GIE-CERBM/ICS
Institut Clinique de la Souris, Illkirch/Strasbourg, France

Sanger Institute
Wellcome Trust Sanger Institute, Hinxton, UK

CNB/CSIC
Centro Nacional de Biotecnología, Madrid, Spain

Fleming
Biomedical Sciences Research Center Al. Fleming, Athens, Greece

OULU
University of Oulu, Oulu, Finland

BIAT
Vetmeduni Vienna, Biomodels Austria, Vienna, Austria

IMG
Institute of Molecular Genetics, Prague, Czech Republic

TAU
Tel Aviv University, Tel Aviv, Israel

NKI
Netherlands Cancer Institute, Netherlands, Amsterdam

www.mousephenotype.org 16 nodes / 13 countries www.infrafrontier.eu

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Reduction
Impact of INFRAFRONTIER on ANIMAL WELFARE
• Experimental design
• Number of animals “saved” (not bred, not born, not • Experimental and control groups, blocks
used) because they are cryopreserved • Adequate number of individuals per experimental
group, according to expected differences, technical
and biological variability
• ~50 animals/year needed to maintain a colony
• Defining the correct number of individuals (sample
size)
• ~ 8.000 mouse lines archived in EMMA‐INFRAFRONTIER

• Up to 400.000 mice per year have been saved, have not

been born

N=5
CALCULATING SAMPLE SIZE
12

10
p=0,08
Let’s assume the parameter we are BEFORE: to cryopreserve ONE mouse line
8 measuring varies +/‐ 20% in the populations
6
NS to be analyzed
4 S
p<0,0001
We required 8‐10 males from the mouse line to be cryopreserved
2

0
Experimental 1:
control experimental 1 experimental 2 Difference between experimental and
control group is about 20%
N=10
We will need N=20 for significant results
12

10
We crossed the males 1:1 once per week during 5‐6 weeks
8 p=0,04 Experimental 2:
6
~NS
Difference between experimental and
S
4
p<0,0001 control group is about 50%
2

0
control experimental 1 experimental 2 It is enough with N=5 for significant results

N=20

12

10

8
p<0,0001
6
S
4 S
p<0,0001
2

0
control experimental 1 experimental 2

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TODAY: to cryopreserve ONE mouse line

Refinement
We require 2‐5 males from the mouse line to be cryopreserved
• Researches must use always the most advanced,
optimized methods and equipments for research
animals aiming to reduce or alliviate, to the
minimum, the harm, pain and discomfort caused by
We obtain oocytes from 2‐5 females for IVF the experimental procedures

• Surgical anesthesia, post‐surgical analgesia

IVF and Cryopreservation methods extremely refined (CARD, Univ. Kumamoto) • Developing new methods, more efficient, allowing
to reach similar conclusions through the use of lesser
From 60‐70 mice to 4‐10 mice in 1 day per ONE line to be cryopreserved number of animals

Refinement Refinement

Uterine embryo transfer NSET Injectable anesthesia Inhalation anesthesia

Non‐surgical embryo transfer (ketamine + xylazine) (isofluorane)

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REFINEMENT
NGS genotyping of CRISPR founder mice

Guías / Recomendaciones ARRIVE

https://arriveguidelines.org/
2010/63/EU
RD53/2013

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New European Directive New European Directive

DIRECTIVE 2010/63/EU OF THE

EUROPEAN PARLIAMENT AND OF
THE COUNCIL of 22 September 2010
on the protection of animals used
for scientific purposes

Mandatory to all EU member states

Must be adopted and transposed to

National regulations by 10 Nov 2012

Came into force on 1 January 2013

Final goal: full replacement of procedures on live animals

Transposition of EU Directive to Spain Transposition of EU Directive to Spain

La Nueva Directiva Europea Summary:

2010/63/UE fue finalmente
traspuesta al ordenamiento
jurídico español a través del Real A set of regulations aiming to permit animal
Decreto 53/2013, de 1 de experimentation ONLY in registered centres,
febrero, por el que se establecen
las normas básicas aplicables by competent personnel, adequately
para la protección de los educated and after obtaining the
animales utilizados en
experimentación y otros fines corresponding authorization
científicos, incluyendo la
docencia, publicado en el B.O.E.
el 8 de febrero de 2013

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New European Directive New European Directive

in Spain
LOCAL LOCAL
committee committee
Project Project
validation validation Habilitated Organ
Project evaluation

Project Competent Project Competent

Research Authority Research Authority
Animal authorization Animal authorization

PERCEPCIÓN SOCIAL de la EXPERIMENTACIÓN

ANIMAL
Comités de ética

•Animales
•RD53/2013, L6/2013, ECC/566/2015
•OEBA – CEEA valida
•Órgano Habilitado evalúa
•Autoridad Competente autoriza
•CEPAFIC coordina, armoniza

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Can we reproduce all experiments

using cells in culture?

Hw do we reproduce the architecture of complex organs?

How do we investigate on vision using cells?

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3D-bioprinted organs/organoids?

On Mice and Men: we share most of the genes

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Animal Models of Human Rare Diseases

Albinism

Presented June 2013

1,2 million signatures collected in EU

They asked for complete ban of Directive 2010/63/EU

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Official Response from EC to STOP VIVISECTION

1. Proposal rejected.
2. It is still premature to ban animal
experimentation because it is still required.
3. Promote alternative methods.
2015

COSCE = Federation of Spanish Scientific Societies

Reaction from Research Community
• Disperse
• Individual actions, non‐coordinated
• Society not receiving adequate information
• Basel Declaration, 2011 (Animal Res. Tomorrow)
• Understanding Animal Research UK and EARA
• Scientific Societies In Spain: FIRST white document on the use of animals in research
• Spain: COSCE, Jan 2014

Feb 2015

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How to “fight” animal activists?

• Without violence
• By pro‐actively providing trusted, serious and
reliable information to Society on why, where,
when and how we use animals in research
• Initiatives on Openness on Animal Research
• First one launched in UK in 2014 (Concordat)
• Second one launched in Spain in 2016
• Two more launched in Portugal and Belgium in UK Concordat on Openness on
Animal Research, launched in 2014
2019
128 organisations have signed so far
• Informative web page in Germany in 2016 May 2022

Transparency in Animal Experimentation Transparency in Animal Experimentation

• The agreement has four points (adapted from British Concordat)

• Agreement promoted by COSCE
• Adapting the UK Concordat to Spain
• With the support & advice from EARA
• Approved by COSCE on 7 June 2016
• Launched on 20 September 2016
• 159 Spanish institutions already adhered
– 1 Speak with clarity on when, how and why we
use animals in research
– 2 Provide adequate information to mass media
and lay public on the conditions in which we do
research projects that require the use of animals
and about the results we derive from them

Mayo de 2022

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Transparencia en Experimentación Animal en España 159

Transparency in Animal Experimentation

– 3 Promote initiatives to generate a better

knowledge and understanding in the society
on the use of animals in research

– 4 Inform annually on the progress of the

agreement and sharing experiences

What type of institutions adhered to the Countries with Transparency Agreements (x9)
Transparency Agreement in Spain?

• 60 Research Centres There are currently nine countries with Transparency agreements: UK (2014), Spain (2016), Portugal (2018), Belgium (2019),
France (2021), Germany (2021), Netherlands (2021), New Zealand (2021) and Switzerland (2022)

• 37 Scientific Societies
• 31 Universities
• 13 Private Companies
• 8 Research Institutions/Organizations
• 3 Science Parks
2014 2016 2018 2019 2021 2021 2021 2021
• 5 Patients Associations
• 2 Hospital >500 institutions adhered world-wide
Total = 159 2022 eara.eu

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Dedicated web page for the Spanish transparency agreement

Practising Transparency

https://cosce.org/acuerdo-de-transparencia/

Número de (usos de) TOTALES de animales por año

Estadísticas del número de usos de
animales en investigación, enseñanza
y otros fines

Año 2020
Fuente: MAPA
Gráficos: Lluís Montoliu (CSIC)

Número de animales Número de USOS de animales

Reducción del -7% desde 2019

https://www.mapa.gob.es/es/ganaderia/temas/produccion-y-mercados-ganaderos/bienestanimal/en-la-investigacion/Informes_y_publicaciones.aspx
Reducción del -9% desde 2018
Fuente: MAPA
https://www.mapa.gob.es/es/ganaderia/temas/produccion-y-mercados-ganaderos/informeestadisticodeanimalesutilizadosen2020_tcm30-580003.pdf Reducción del -46% desde 2009

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Number os animal uses in Spain between 2009 and 2020 Número de (usos de) animales por año
(primer uso / reutilización)

*
1.403.290 animal uses in 2009
761.012 animal uses in 2020
- 46%
Source: MAPA Fuente: MAPA

Severidad de los Procedimientos (2020)

FINALIDAD de (usos de) animales por año

La gran mayoría de animales (78%) se destina a investigación

85% de procedimientos son leves o moderados
Fuente: MAPA Fuente: MAPA 10% de procedimientos son severos

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Severidad de los Procedimientos (2014-2020)

Especies animales utilizadas (2020)

Fuente: MAPA
Porcentajes muy similares
Fuente: MAPA 86% de procedimientos son leves o moderados 63% de los usos de animales corresponde a “roedores”
7% de procedimientos son severos

Fuente: MAPA Especies animales utilizadas (2009-2020)

Institutional Statements on the Use of Research Animals

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Institutional Statements on the Use of Research Animals Institutional Statements on the Use of Research Animals

www.csic.es

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1st July 2021: First International Day on Transparency on Animal Research

SEBBM BOARD21: Jornada Internacional por la Transparencia en Experimentación Animal

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Lluís Montoliu
montoliu@cnb.csic.es
wwwuser.cnb.csic.es/montoliu

Comité de Ética del CSIC

comitedeetica@csic.es
www.csic.es/es/el-csic/etica

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